Toxicity of pesticide cocktails in amphibian larvae: understanding the impact of agricultural activity on aquatic ecosystems in the Salado River basin, Argentina.

Aquatic communities are increasingly exposed to complex mixtures of contaminants, mainly pesticides due to the impact of agricultural activity. The aim of this study was to evaluate the toxicity of an eight-pesticide cocktail on larvae of the South American common toad, Rinella arenarum. The cocktail represents a realistic mixture of insecticides (cypermethrin, chlorpyrifos and lambda-cyhalothrin), herbicides (glyphosate, glufosinate ammonium, prometryn and metolachlor), and a fungicide (pyraclostrobin) previously found in aquatic organisms (Prochilodus lineatus) from the Salado River Basin, an area with strong agricultural pressure. Computational simulations through the Density Functional Tight-Binding method indicated a strong spontaneous trend toward the formation of the cocktail, suggesting that it may act as a novel xenobiotic entity in the environment. The cocktail effects were evaluated in early-developing and premetamorphic larvae, at feasible concentrations found in real scenarios. The mixture led to high mortality and teratogenicity in early-developing larvae. Premetamorphic larvae showed endocrine disruption, oxidative stress, and impairments in detoxification and hepatic functioning. Neurotoxicity, genotoxicity, cardiotoxicity and high mortality under stress conditions were also observed in exposed larvae. This novel evaluation highlights the ecotoxicological risk for aquatic organisms exposed to complex mixtures and underscores the need to consider cocktail effects in studies regarding ecosystems health.

High toxicity of agro-industrial wastewater on aquatic fauna of a South American stream: Mortality of aquatic turtles and amphibian tadpoles as bioindicators of environmental health.

The aim of this study was to characterize an aquatic system of Santa Fe province (Argentina) receiving wastewater from agro-industrial activities (mainly dairy) by in situ assessment (fauna mortality, physicochemical, microbiological, and pesticide residues measurement), and ecotoxicity bioassays on amphibian tadpoles. Water and sediment samples were obtained from the Los Troncos Stream (LTS), previous to the confluence with the "San Carlos" drainage channel (SCC), and from the SCC. Biological parameters (mortality and sublethal biomarkers) were used to evaluate ecotoxicity during 10-day exposure of Rhinella arenarum tadpoles to LTS and SCC samples. Nine pesticides were detected in both LTS and SCC. Chemical and biochemical oxygen demand, ammonia, and coliform count recorded in SCC greatly exceeded limits for aquatic life protection. At SCC and LTS after the confluence with SCC, numerous dying and dead aquatic turtles (Phrynops hilarii) were recorded. In the ecotoxicity assessment, no mortality of tadpoles was observed in LTS treatment, whereas total mortality (100%) was observed in SCC treatments in dilution higher than 50% of water and sediment. For SCC, median lethal concentration and the 95% confidence limits was 18.30% (14.71-22.77) at 24 h; lowest-observed and no-observed effect concentrations were 12.5% and 6.25%, respectively. Oxidative stress and neurotoxicity were observed in tadpoles exposed to 25% SCC dilution treatment. In addition, there was a large genotoxic effect (micronuclei test) in all sublethal SCC dilution treatments (6.25%, 12.5%, and 25%). These results alert about the high environmental quality deterioration and high ecotoxicity for aquatic fauna of aquatic ecosystems affected by agro-industrial wastewater. PRACTITIONER POINTS: Great mortality of turtles was observed in a basin with a high load of agro-industrial wastewater. San Carlos Channel (SCC), where effluents are spilled, is environmentally deteriorated. The water-sediment matrix of SCC caused 100% lethality in tadpoles. SCC dilutions caused neurotoxicity, oxidative stress, and genotoxicity on tadpoles.

Ecotoxicological Characterization of Lithium as a "Timebomb" in Aquatic Systems: Tadpoles of the South American Toad Rhinella arenarum (Hensel, 1867) as Model Organisms.

The aim of this study was to evaluate the acute lethality and chronic sublethal effects of lithium (Li) on Rhinella arenarum tadpoles as model organisms. First a 96 h toxicity assay was performed by exposing tadpoles to Li concentrations from 44.08 to 412.5 mg L-1 to estimate the mortality, and lethal and sublethal effects. Another bioassay was carried out by exposing tadpoles to two environmentally relevant Li concentrations (2.5 and 20 mg L-1) for one and two weeks. The sublethal effects of Li on tadpoles were evaluated by analyzing biochemical, genotoxic, and physiological biomarkers. The mortality in Li-exposed tadpoles increased over time. The median lethal concentration (LC50) ranged from 319.52 (281.21-363.05) mg L-1 at 48 h to 66.92 (52.76-84.89) mg L-1 at 96 h. Exposure to Li at 2.5 and 20 mg L-1 induced alterations in enzymes related to detoxification, antioxidant, and hepatic mechanisms, endocrine disruption of thyroid hormones, genotoxicity, and effects on the physiology of the heart and gastrointestinal systems. Tadpoles exposed to the highest concentration in the chronic bioassay (20 mg L-1 Li), which is the concentration commonly recorded in Li mining sites, showed significant mortality after one week of exposure. These results warn about the high ecotoxicological risk of Li as a contaminant of emerging concern for amphibians.

Effect of glyphosate and ciprofloxacin exposure on enteric bacteria of tadpoles / Efecto de la exposición a glifosato y ciprofloxacina en bacterias entéricasde renacuajos

Abstract The high load of agrochemicals and antibiotics present in agricultural aquatic environments represents a risk for wildlife. Since enteric bacteria, which play a key role in the physiological functioning of their hosts, are sensitive to a wide variety of pollutants, their study allows to evaluate the health of organisms. This study aimed to evaluate the effects of commercial formulations of a glyphosate-based herbicide (GBH) and the antibiotic ciprofloxacin (CIP), individually and in mixture, on the bacterial diversity of the intestinal content of common toad (Rhinella arenarum) tadpoles. The diversity of cultivable fast-growing bacteria with low nutritional requirements was evaluated using classic microbiological tests and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification. Bacterial diversity varied among treatments. Taxa diversity increased in the GBH-treated group but decreased in the CIP-treated group. Remarkably, Yersinia spp. and Proteus spp. were only found in the GBH-treated group. The prevalence of Klebsiella spp. and Pseudomonas spp. decreased in the intestinal microbiota of the GBH-CIP-treated group. To our knowledge, this is the first report on the alteration of cultivable enteric bacteria of autochthonous tadpoles due to two pollutants of emerging concern. Our results demonstrate that R. arenarum tadpoles can be used as non-conventional model organisms for environmental pollution monitoring. Our preliminary findings would contribute to understanding how the presence of GBH and CIP in freshwaters may represent a threat to wildlife and human health by causing enteric dysbiosis of part of the bacterial community.

Resumen La alta carga de agroquímicos y antibióticos en los ambientes acuáticos y los agroe-cosistemas representa un riesgo para la vida silvestre. Dado que la microbiota intestinal juega un papel fundamental en el funcionamiento de su hospedador y es sensible a una amplia variedad de contaminantes, su estudio permite evaluar la salud de los organismos. En este trabajo estudiamos los efectos de formulaciones comerciales de un herbicida a base de glifosato (GBH) y del antibiótico ciprofloxacina (CIP), por separado y en mezcla, sobre la diversidad de bacterias intestinales de renacuajos del sapo común (Rhinella arenarum). El estudio de la diversidad de bacterias entéricas cultivables de rápido crecimiento y bajo requerimiento nutricional se llevó a cabo utilizando pruebas microbiológicas clásicas e identificación por espectrometría de masas de tiempo de vuelo por desorción/ionización láser asistida por matriz (MALDI-TOF). La microbiota entérica fue diferente según el tratamiento. El GBH indujo un aumento de la diversidad bacteriana, mientras que la CIP produjo una reducción. Entre estos cambios, destaca la presencia de Yersinia spp. y Proteus spp. solo en el tratamiento con GBH. Además, en el tratamiento GBH-CIP se encontró una disminución en la prevalencia de Klebsiella spp. y Pseudomonas spp. en la microbiota intestinal de los renacuajos. Este es el primer informe sobre la alteración del contenido bacteriano intestinal de renacuajos de R. arenarum producido por dos contaminantes emergentes de preocupación. Demostramos que el renacuajo del sapo común se puede utilizar como un organismo modelo no convencional para el monitoreo de la contaminación ambiental. Estos hallazgos constituyen el primer paso para comprender cómo la presencia de GBH y CIP en aguas dulces puede representar una amenaza para la vida silvestre y la salud humana a través de la disbiosis entérica asociada al efecto sobre la comunidad bacteriana.

Water and sediment quality of an agro-industrial area through different approaches: Physicochemical parameters and ecotoxicity with amphibians.

Amphibians are subject to several stressors in the aquatic and terrestrial environments, and human activities have profoundly impacted this vertebrate group. The aim of the present study was to analyze physicochemical parameters, metals and pesticide residues, and the toxicity of water and sediment samples from an environment with high agricultural activity (S1: Salto stream; S2: drainage channel downstream from S1) by means of bioassays using Rhinella arenarum (Amphibia: Anura) larvae. Metals and pesticides were analyzed in water and sediment samples by fluorescence spectrometer of X-ray by total reflection and ultra-high-performance liquid chromatography-MS/MS, respectively. For lethality bioassays, 10 larvae (in triplicate) were exposed for 504 h to water and sediment samples. Also, 50 larvae were exposed for 96 h (in triplicate) to water and sediment samples for the evaluation of biomarkers of neurotoxicity, oxidative stress, and genotoxicity. Twenty-six different pesticides (mainly herbicides) were detected in both sites, and Cu, Zn, and Pb exceeded the limit for protection of aquatic life. Lethality was observed in larvae exposed to water and sediment samples from both sites at chronic exposure. Oxidative stress was observed in larvae exposed to both sites. In larvae exposed to samples from S1, alterations in the neurotoxicity biomarkers were observed. These results alert about the degradation of the sites and highlight the need to monitor and control the use of pesticides. PRACTITIONER POINTS: Twenty-six pesticides were detected in water and sediment from Salto stream basin. Significant mortality was observed in larvae exposed to samples from all sites. Sublethal effects were observed mainly in larvae exposed to samples from Salto stream. The degraded quality can be associated with the agricultural activities of the area.

Cocktails of pesticide residues in Prochilodus lineatus fish of the Salado River (South America): First record of high concentrations of polar herbicides.

Muscle and viscera (gills-liver) of the fish Prochilodus lineatus were obtained from four sites of lower course of Salado river and one site at Santa Fe river near to its confluence with Salado river from Santa Fe (Argentina) between December 2021 and February 2022. Sediment samples were also obtained from the same sites. All samples were analyzed for pesticide residues following the QuEChERS method to quantify 136 compounds by UHPLC-ESI-MS/MS and GC-EI-MS/MS. Overall, muscle fish tissue showed very high concentrations (maximum concentrations detected) of the insecticide cypermethrin (204 µg/kg), polar herbicides (glyphosate; 187 µg/kg and its degradation product (aminomethylphosphonic acid) AMPA; 3116 µg/kg, and glufosinate-ammonium; 677 µg/kg), and the fungicide pyraclostrobin (50 µg/kg). In viscera samples, high values of cypermethrin (506 µg/kg), chlorpyrifos (78 µg/kg), and lambdacyhalothrin (73 µg/kg) were the main pesticides found. Mean residues concentrations detected among sites were not significantly different neither in muscle nor viscera of P. lineatus in most of the cases. Exceptionally, the southernmost studied site of the Lower Salado river showed significant differences in concentration of residues found in muscle, due to high concentrations of glyphosate and glufosinate-amonium (KW = 11.879 and KW = 13.013, respectively, P < 0.05). Other norther Lower Salado river site showed significant higher AMPA concentration in fish viscera than in the rest of the studied sites (KW = 12.86 P < 0.05). Some sediment samples showed low levels of herbicides such as glyphosate (24 µg/kg) and fungicides. However, the world highest levels of polar herbicides were recorded in fish muscle. The results of this study highlight the need for periodic monitoring due to the high concentration of pesticides and its potential risk in a very important commercial freshwater fish from Argentina, which is consumed locally and exported to other countries for human consumption.

Effect of glyphosate and ciprofloxacin exposure on enteric bacteria of tadpoles.

The high load of agrochemicals and antibiotics present in agricultural aquatic environments represents a risk for wildlife. Since enteric bacteria, which play a key role in the physiological functioning of their hosts, are sensitive to a wide variety of pollutants, their study allows to evaluate the health of organisms. This study aimed to evaluate the effects of commercial formulations of a glyphosate-based herbicide (GBH) and the antibiotic ciprofloxacin (CIP), individually and in mixture, on the bacterial diversity of the intestinal content of common toad (Rhinella arenarum) tadpoles. The diversity of cultivable fast-growing bacteria with low nutritional requirements was evaluated using classic microbiological tests and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification. Bacterial diversity varied among treatments. Taxa diversity increased in the GBH-treated group but decreased in the CIP-treated group. Remarkably, Yersinia spp. and Proteus spp. were only found in the GBH-treated group. The prevalence of Klebsiella spp. and Pseudomonas spp. decreased in the intestinal microbiota of the GBH-CIP-treated group. To our knowledge, this is the first report on the alteration of cultivable enteric bacteria of autochthonous tadpoles due to two pollutants of emerging concern. Our results demonstrate that R. arenarum tadpoles can be used as non-conventional model organisms for environmental pollution monitoring. Our preliminary findings would contribute to understanding how the presence of GBH and CIP in freshwaters may represent a threat to wildlife and human health by causing enteric dysbiosis of part of the bacterial community.

Environmental quality and ecotoxicity of sediments from the lower Salado River basin (Santa Fe, Argentina) on amphibian larvae.

The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the aim was to evaluate the quality of three sampling sites that belong to the Salado River basin (S1: Cululú stream; S2: Salado River, at Esperanza City, S3: Salado River at Santo Tomé City) based on physicochemical parameters, metals and pesticides analyses and ecotoxicity on Rhinella arenarum larvae. R. arenarum larvae (Gosner Stage -GS- 25) were chronically exposed (504h) to complex matrixes of surface water and sediment samples of each site for the determination of the survival rate. Biomarkers of oxidative stress, neurotoxicity and genotoxicity were analyzed in R. arenarum larvae (GS. 25) after exposure (96h) to the complex matrix of water and sediment. The water quality index showed a marginal quality for all sites, influenced mainly by low dissolved oxygen, high total suspended solid, phosphate, nitrite, conductivity, Pb, Cr and Cu levels. Metal concentrations were higher in sediment than in water samples (˜34-35000 times). In total, thirty different pesticides were detected in all water and sediment samples, S1 presented the greatest variety (26). Glyphosate and AMPA were detected in sediments from all sites, being higher in S3. N,N-Diethyl-meta-toluamide (DEET) and atrazine were detected in all water samples. Greatest mortality was observed in larvae exposed to samples from S1 from 288h (43.3%), reaching a maximum value of 50% at 408h. Oxidative stress and genotoxicity were observed in larvae exposed to S1 and S3 matrix samples. Neurotoxicity was observed in larvae exposed to all matrix samples. The integrated biomarker response index showed that larvae exposed to S1 and S3 were the most affected. According to the physicochemical data and the ecotoxicity assessment, this important river basin is significantly degraded and may represent a risk to aquatic biota, especially for R. arenarum larvae.

Effect of microplastics on the activity of carboxylesterase and phosphatase enzymes in Scinax squalirostris tadpoles.

Microplastics (MPs) are critical emerging pollutants around the world. There is a growing interest in the effects of MP ingestion, non-digestion, and toxicity on aquatic organisms. Amphibian tadpoles are the vertebrate group that has received the least attention regarding this issue. The aim of the present study was to determine the ingestion of polyethylene MPs by Scinax squalirostris tadpoles by atomic force microscopy (AFM) and to evaluate the activities of carboxylesterase (CbE, using 4-naphthyl butyrate-NB-, and 1-naphthyl acetate -NA- as substrates) and alkaline phosphatase (ALP) under MP exposure. Enzyme activities were analyzed spectrophotometrically at 2 and 10 days of exposure. Tadpoles were exposed to two different treatments during 10 days: a negative control (CO, dechlorinated water) and MP (60 mg L-1). AFM images of the digestive contents of tadpoles revealed the presence of MPs. After 10 days of MP exposure, CbE (NB) activity was significantly higher and CbE (NA) activity was significantly lower in MP treatments than in controls. ALP activity decreased in MP treatments after 2 and 10 days of exposure. The detection of MP particles in the intestinal contents and the effects on metabolic enzymes in a common frog species evidenced the potential health risk of MP to aquatic vertebrates. Thus, the differential response in enzymes and substrates demonstrate the need for considering the complex effects of contaminants and nutrients on ecosystems for ecotoxicological risk characterization.

The integrated biomarker response in three anuran species larvae at sublethal concentrations of cypermethrin, chlorpyrifos, glyphosate, and glufosinate-ammonium.

The aim of the present study was to evaluate the response in larvae of the anuran species Rhinella arenarum, Rhinella dorbignyi and Odontophrynus americanus exposed to glyphosate (GLY, 2.5 mg L-1), cypermethrin (CYP, 0.013 mg L-1), chlorpyrifos (CP, 0.1 mg L-1) and glufosinate-ammonium (GLU, 15 mg L-1) using two behavioral endpoints: mean speed (MS) and total distance moved (TD); and two enzymatic biomarkers: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In order to assess a global response and to determine the most sensitive species, an integrated biomarker response (IBR) index was calculated. Behavioral biomarkers were tested at 1 and 60 min, and the enzymes at 60 min after exposure. The results showed that: (1) there were statistical differences between species in a series of responses in swimming behavior, and cholinesterase activities within the first-hour of exposure to CYP, GLY, and CP at environmentally relevant concentrations (ERC); (2) IBR determined that Rhinella species were the most sensitive of the species tested and (3) IBR provided a comprehensive assessment of the health status of species exposed to ERC of a wide variety of agrochemicals globally and frequently used.

Morphological and histological abnormalities of the neotropical toad, Rhinella arenarum (Anura: Bufonidae) larvae exposed to dexamethasone.

Dexamethasone (DEX) is a glucocorticoid highly effective as an anti-inflammatory, immunosuppressant and decongestant drug. In the present study, a preliminary acute toxicity test was assayed in order to determinate DEX median-lethal, lowest-observed-effect and the no-observed-effect concentrations (LC50, LOEC and NOEC, respectively) on the common toad embryos (Rhinella arenarum). Also, morphological and histological abnormalities from five body larval regions, liver melanomacrophages (MM) and glutathione S-transferase (GST) activity were evaluated in the toad larvae to characterize the chronic sublethal effects of DEX (1-1,000 µg L-L). Results of the acute test showed that the LC50 of DEX at 96 h of exposure for the toad embryos (GS 18-20) was 10.720 mg L-g, and the LOEC was 1 µg L-g. In the chronic assay, the larval development and body length were significantly affected. DEX exposition also induced teratogenic effects. Most frequent external abnormalities observed in DEX-treated larvae included abdominal edema and swollen body, abnormal gut coiling and visceral congestion. Intestinal dysplasia was recurrent in cross-section of all DEX-treated larvae. Neural, conjunctive and renal epithelial cells were also affected. Significant increase in liver MM number and size, and GST activity levels were also registered in DEX treatments with respect to controls. The evaluation of a variety of biomarkers provided clear evidence of toad larvae sensitivity to DEX, and the ecotoxicological risk of these pharmaceuticals, commonly found in different water bodies worldwide on aquatic animals.

Can agroecological management increase functional diversity of birds in rice fields? / ¿Puede el manejo agroecológico incrementar la diversidad funcional de aves en campos de arroz?

Introduction: Recognition of the variety of ecosystem services that biodiversity performs in agroecosystems is one of the basic principles of agroecology. Because indices of functional diversity may be directly related with ecosystem services, an assessment of functional diversity can be useful for evaluating ecosystem services provided under agroecological management. Objective: We compared functional diversity of birds found in rice fields under conventional and agroecological management in the rice zone of Santa Fe Province, Argentina. Our objective was to determine whether agroecological management of rice is associated with a higher functional diversity and a different functional composition of birds than conventional management. Methods: We surveyed birds eight times, roughly every 15 days between November 2017 and March 2018, from beginning of sowing to before harvest, in both types of rice fields. Birds were sampled by a combined technique of line transects and point counts at four sites in each type of management. We calculated indices of functional diversity and composition based on morphologic and trophic attributes of birds detected in each type of field. Results: Functional richness, divergence and dispersion were higher under agroecological management. Only differences in functional richness between managements reflected differences in species richness. Community-level weighted means of trait values by sample varied between management types. An insectivorous diet, pursuit as a foraging method, and air and shrubs as foraging substrates were traits best represented under agroecological management. Conclusions: Our results suggest that agroecological management of rice crops is related with a higher functional diversity of birds than conventional practices, suggesting that agroecological management may enhance the provision of ecosystem services by birds in rice agroecosystems.

Introducción: El reconocimiento de la variedad de servicios ecosistémicos de la biodiversidad en los agroecosistemas es uno de los principios básicos de la agroecología. Debido a que los índices de diversidad funcional pueden estar directamente relacionados con los servicios ecosistémicos, la evaluación de la diversidad funcional representa una estrategia adecuada para evaluar estos servicios en agroecosistemas bajo manejo agroecológico. Objetivo: Se comparó la diversidad funcional de aves entre arroceras bajo manejo convencional y agroecológico en la zona arrocera de la provincia de Santa Fe, Argentina. El objetivo fue determinar si el manejo agroecológico del arroz se asocia con una mayor diversidad funcional y con una diferente composición funcional de aves en relación con el manejo convencional. Métodos: Se muestrearon las aves mediante 8 conteos repetidos aproximadamente cada 15 días entre noviembre 2017 y marzo 2018, desde el comienzo de la siembra hasta antes de la cosecha, en ambos tipos de arroceras. Los muestreos fueron realizados mediante una técnica mixta combinando transectas de línea y puntos de conteo en cuatro sitios por tipo de manejo. Se calcularon índices de diversidad y composición funcional con base en atributos morfológicos y tróficos de las aves registradas. Resultados: La riqueza, la divergencia y la dispersión funcional fueron más altos bajo manejo agroecológico. Solamente en el caso de la riqueza funcional las diferencias reflejaron únicamente la variación de la riqueza de especies. La media ponderada a nivel de ensamble de los valores de los rasgos por muestra varió entre los tipos de manejo, estando la dieta insectívora, la persecución como método de forrajeo, y el aire y los arbustos como sustrato de forrajeo mejor representados bajo manejo agroecológico. Conclusión: Los resultados sugirieron que el manejo agroecológico del arroz se relaciona con una mayor diversidad funcional de aves que las prácticas de manejo convencional, sugiriendo que el manejo agroecológico puede mejorar la provisión de servicios ecosistémicos por aves en cultivos de arroz.

Comparative Toxicity of Two Different Dimethoate Formulations in the Common Toad (Rhinella arenarum) Tadpoles.

Dimethoate (D) are among the most commonly used organophosphates insecticides in the world. To evaluate the toxicity of two D formulations were selected as test organisms tadpoles of Rhinella arenarum. This toad species has an extensive neotropical distribution and is easy to handle and acclimate to laboratory conditions. The tadpoles were exposed in an acute assay for 48 h to D soluble concentrates (DSC) and emulsifiable concentrates (DEC). The 48 h-LC50 (95% confidence limits) value of DSC was 57.46 mg L-1 (40.52-81.43) and to DEC was 12.76 mg L-1 (10.39-15.68). These differences in toxicity were statistically significant (p < 0.05). In both formulations, acetylcholinesterase), carboxylesterase, and glutathione-S-transferases enzyme activities varied significantly respect to those of control group (p < 0.05). The DEC formulation was the most toxic. These results would allow the assessment and characterization of potential ecological risks following the application of those formulations.

First evaluation of novel potential synergistic effects of glyphosate and arsenic mixture on Rhinella arenarum (Anura: Bufonidae) tadpoles.

The toxicity of glyphosate-based herbicide (GBH) and arsenite (As(III)) as individual toxicants and in mixture (50:50 v/v, GBH-As(III)) was determined in Rhinella arenarum tadpoles during acute (48 h) and chronic assays (22 days). In both types of assays, the levels of enzymatic activity [Acetylcholinesterase (AChE), Carboxylesterase (CbE), and Glutathione S-transferase (GST)] and the levels of thyroid hormones (triiodothyronine; T3 and thyroxine; T4) were examined. Additionally, the mitotic index (MI) of red blood cells (RBCs) and DNA damage index were calculated for the chronic assay. The results showed that the LC50 values at 48 h were 45.95 mg/L for GBH, 37.32 mg/L for As(III), and 30.31 mg/L for GBH-As(III) (with similar NOEC = 10 mg/L and LOEC = 20 mg/L between the three treatments). In the acute assay, Marking's additive index (S = 2.72) indicated synergistic toxicity for GBH-As(III). In larvae treated with GBH and As(III) at the NOEC-48h (10 mg/L), AChE activity increased by 36.25% and 33.05% respectively, CbE activity increased by 22.25% and 39.05 % respectively, and GST activity increased by 46.75% with the individual treatment with GBH and by 131.65 % with the GBH-As(III) mixture. Larvae exposed to the GBH-As(III) mixture also showed increased levels of T4 (25.67 %). In the chronic assay at NOEC-48h/8 (1.25 mg/L), As(III) and GBH-As(III) inhibited AChE activity (by 39.46 % and 35.65%, respectively), but did not alter CbE activity. In addition, As(III) highly increased (93.7 %) GST activity. GBH-As(III) increased T3 (97.34%) and T4 (540.93%) levels. Finally, GBH-As(III) increased the MI of RBCs and DNA damage. This study demonstrated strong synergistic toxicity of the GBH-As(III) mixture, negatively altering antioxidant systems and thyroid hormone levels, with consequences on RBC proliferation and DNA damage in treated R. arenarum tadpoles.

Effect of traffic noise on Scinax nasicus advertisement call (Amphibia, Anura) / Efecto del ruído del tráfico en la vocalización de machos de Scinax nasicus (Amphibia, Anura)

El aumento de los sonidos de origen antropogénico, como los ruidos del tráfico, contribuyen a la contaminación acústica, que produce un efecto nocivo en el canto de los vertebrados. Comparamos la vocalización de machos de Scinax nasicus (Cope, 1862) en ambientes naturales (como referencia o control, Sitio A) y sitios afectados por ruidos de tráfico (Sitio B). La estructura de la vocalización se registró y amplificó en sonogramas (software Raven Pro 1.5). Se midieron siete variables de su vocalización: duración (s), número de notas, número de pulsos por nota, frecuencia máxima, mínima y dominante (kHz) y amplitud (dB). Además, en cada sitio se midió el ruido de fondo (frecuencia fundamental, la F0 y amplitud, dB). La amplitud del ruido de fondo alcanzó valores más altos (68.02 dB) en el Sitio B, en el Sitio A fue menor (34.81 dB). Por lo tanto, el F0 en el Sitio A fue de 6.28 kHz y en el Sitio B fue de 4.15 kHz. Las vocalizaciones de esta rana en el ambiente con ruido de tráfico (Sitio B) se caracterizaron por menor duración (s) y número de pulsos por nota, mayor amplitud (dB) y frecuencias máximas y dominantes más altas (kHz), baja frecuencia mínima en comparación con el ambiente control (Sitio A). Nuestro estudio resaltó que los machos de S. nasicus cambian su estructura vocal en estanques de ruido de tráfico, principalmente por ajuste vocal de sus frecuencias y amplitud para contrarrestar el efecto de enmascaramiento del ruido. Finalmente, el monitoreo acústico de anuros en ambientes ruidosos debe considerar el solapamiento espacial, temporal y espectral entre ruido acústico específico y el comportamiento de la especie.(AU)

Increased anthropogenic-made sounds such as traffic noises contribute to acoustic pollution, which produces deleterious effect on song-vertebrates. We compared the advertisement call of Scinax nasicus (Cope, 1862) males in natural (as a reference or control, Site A) and Sites affected by traffic noises (Site B). Call structure was recorded and it was amplified in sonograms (software Raven Pro 1.5). Seven variables were measured on its advertisement call: duration (s), number of notes, number of pulses per note, maximum and minimum frequency (kHz), dominant frequency (kHz) and amplitude (dB). In addition, at each Site the background noise (the fundamental frequency, F0 and amplitude, dB) was measured. The amplitude of background noise reached higher values (68.02 dB) in Site B, while in Site A was lower (34.81 dB). Thus, the F0 in Site A was 6.28 kHz and in Site B it was 4.15 kHz. Frog call in noisy environment (Site B) were characterized by lesser duration (s) and number of pulses per note, higher maximum and dominant frequencies (kHz), lower minimum frequencies, and amplitude (dB) when compared with control environment (Site A). Our study highlights, that S. nasicus males shift their vocal structure in traffic noisy ponds, mainly by vocal adjust of their frequencies and amplitude to counteract masking effect. Finally, acoustic monitoring of anurans on noise environments should be considering the spatial, temporal and spectral overlap between noise and species-specific acoustic behaviour.(AU)

Effect of traffic noise on Scinax nasicus advertisement call (Amphibia, Anura) / Efecto del ruído del tráfico en la vocalización de machos de Scinax nasicus (Amphibia, Anura)

El aumento de los sonidos de origen antropogénico, como los ruidos del tráfico, contribuyen a la contaminación acústica, que produce un efecto nocivo en el canto de los vertebrados. Comparamos la vocalización de machos de Scinax nasicus (Cope, 1862) en ambientes naturales (como referencia o control, Sitio A) y sitios afectados por ruidos de tráfico (Sitio B). La estructura de la vocalización se registró y amplificó en sonogramas (software Raven Pro 1.5). Se midieron siete variables de su vocalización: duración (s), número de notas, número de pulsos por nota, frecuencia máxima, mínima y dominante (kHz) y amplitud (dB). Además, en cada sitio se midió el ruido de fondo (frecuencia fundamental, la F0 y amplitud, dB). La amplitud del ruido de fondo alcanzó valores más altos (68.02 dB) en el Sitio B, en el Sitio A fue menor (34.81 dB). Por lo tanto, el F0 en el Sitio A fue de 6.28 kHz y en el Sitio B fue de 4.15 kHz. Las vocalizaciones de esta rana en el ambiente con ruido de tráfico (Sitio B) se caracterizaron por menor duración (s) y número de pulsos por nota, mayor amplitud (dB) y frecuencias máximas y dominantes más altas (kHz), baja frecuencia mínima en comparación con el ambiente control (Sitio A). Nuestro estudio resaltó que los machos de S. nasicus cambian su estructura vocal en estanques de ruido de tráfico, principalmente por ajuste vocal de sus frecuencias y amplitud para contrarrestar el efecto de enmascaramiento del ruido. Finalmente, el monitoreo acústico de anuros en ambientes ruidosos debe considerar el solapamiento espacial, temporal y espectral entre ruido acústico específico y el comportamiento de la especie.

Increased anthropogenic-made sounds such as traffic noises contribute to acoustic pollution, which produces deleterious effect on song-vertebrates. We compared the advertisement call of Scinax nasicus (Cope, 1862) males in natural (as a reference or control, Site A) and Sites affected by traffic noises (Site B). Call structure was recorded and it was amplified in sonograms (software Raven Pro 1.5). Seven variables were measured on its advertisement call: duration (s), number of notes, number of pulses per note, maximum and minimum frequency (kHz), dominant frequency (kHz) and amplitude (dB). In addition, at each Site the background noise (the fundamental frequency, F0 and amplitude, dB) was measured. The amplitude of background noise reached higher values (68.02 dB) in Site B, while in Site A was lower (34.81 dB). Thus, the F0 in Site A was 6.28 kHz and in Site B it was 4.15 kHz. Frog call in noisy environment (Site B) were characterized by lesser duration (s) and number of pulses per note, higher maximum and dominant frequencies (kHz), lower minimum frequencies, and amplitude (dB) when compared with control environment (Site A). Our study highlights, that S. nasicus males shift their vocal structure in traffic noisy ponds, mainly by vocal adjust of their frequencies and amplitude to counteract masking effect. Finally, acoustic monitoring of anurans on noise environments should be considering the spatial, temporal and spectral overlap between noise and species-specific acoustic behaviour.

Effect of traffic noise on Scinax nasicus advertisement call (Amphibia, Anura)

RESUMEN El aumento de los sonidos de origen antropogénico, como los ruidos del tráfico, contribuyen a la contaminación acústica, que produce un efecto nocivo en el canto de los vertebrados. Comparamos la vocalización de machos de Scinax nasicus (Cope, 1862) en ambientes naturales (como referencia o control, Sitio A) y sitios afectados por ruidos de tráfico (Sitio B). La estructura de la vocalización se registró y amplificó en sonogramas (software Raven Pro 1.5). Se midieron siete variables de su vocalización: duración (s), número de notas, número de pulsos por nota, frecuencia máxima, mínima y dominante (kHz) y amplitud (dB). Además, en cada sitio se midió el ruido de fondo (frecuencia fundamental, la F0 y amplitud, dB). La amplitud del ruido de fondo alcanzó valores más altos (68.02 dB) en el Sitio B, en el Sitio A fue menor (34.81 dB). Por lo tanto, el F0 en el Sitio A fue de 6.28 kHz y en el Sitio B fue de 4.15 kHz. Las vocalizaciones de esta rana en el ambiente con ruido de tráfico (Sitio B) se caracterizaron por menor duración (s) y número de pulsos por nota, mayor amplitud (dB) y frecuencias máximas y dominantes más altas (kHz), baja frecuencia mínima en comparación con el ambiente control (Sitio A). Nuestro estudio resaltó que los machos de S. nasicus cambian su estructura vocal en estanques de ruido de tráfico, principalmente por ajuste vocal de sus frecuencias y amplitud para contrarrestar el efecto de enmascaramiento del ruido. Finalmente, el monitoreo acústico de anuros en ambientes ruidosos debe considerar el solapamiento espacial, temporal y espectral entre ruido acústico específico y el comportamiento de la especie.

ABSTRACT Increased anthropogenic-made sounds such as traffic noises contribute to acoustic pollution, which produces deleterious effect on song-vertebrates. We compared the advertisement call of Scinax nasicus (Cope, 1862) males in natural (as a reference or control, Site A) and Sites affected by traffic noises (Site B). Call structure was recorded and it was amplified in sonograms (software Raven Pro 1.5). Seven variables were measured on its advertisement call: duration (s), number of notes, number of pulses per note, maximum and minimum frequency (kHz), dominant frequency (kHz) and amplitude (dB). In addition, at each Site the background noise (the fundamental frequency, F0 and amplitude, dB) was measured. The amplitude of background noise reached higher values (68.02 dB) in Site B, while in Site A was lower (34.81 dB). Thus, the F0 in Site A was 6.28 kHz and in Site B it was 4.15 kHz. Frog call in noisy environment (Site B) were characterized by lesser duration (s) and number of pulses per note, higher maximum and dominant frequencies (kHz), lower minimum frequencies, and amplitude (dB) when compared with control environment (Site A). Our study highlights, that S. nasicus males shift their vocal structure in traffic noisy ponds, mainly by vocal adjust of their frequencies and amplitude to counteract masking effect. Finally, acoustic monitoring of anurans on noise environments should be considering the spatial, temporal and spectral overlap between noise and species-specific acoustic behaviour.

Blood biomarkers of common toad Rhinella arenarum following chlorpyrifos dermal exposure.

Chlorpyrifos (CPF) is a broad spectrum pesticide commonly used for insect control, has great affinity for lipids and is thus a potential for bioaccumulation in aquatic organisms. The aim of this study was to evaluate the toxicity of CPF using the common toad Rhinella arenarum via dermal uptake in plastic bucket to simulate their natural exposition in ponds. R. arenarum toads were exposed individually to solutions containing a nominal concentration of a commercial formulation of CPF insecticide (5 and 10 mg/L). Different enzyme biomarkers (BChE: butyrylcholinesterase, CbE: carboxylesterase, and CAT: catalase) were measured in blood tissue after exposition. The capacity of pyridine-2-aldoxime methochloride (2-PAM) to reverse OP-inhibited plasma BChE and the ratio of heterophils and lymphocytes (H/L) as hematological indicators of stress were also determined. The normal values of plasma B-sterases (BChE and CbE) were highly inhibited (until ≈ 70%) in toads 48 h after exposure to CPF. The results indicate that 2-PAM produced BChE reactivation as well. The activity of CAT was also inducted for dermal exposure at more than double of that in the control toads (CPF; 5 mg/L). H/L ratios did not reveal a significantly increased stress. The study suggests that CPF via dermal uptake induced neurotoxicity and oxidative stress in the common toad R. areanum. Thus, some blood biomarkers employed in our study (i.e. BChE, CbE, 2-PAM, and CAT) might be used as predictors in health and ecological risk assessment of amphibian populations exposed to CPF.

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two ß-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000µgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10µgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000µgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10µgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Plasma retinoids concentration in Leptodactylus chaquensis (Amphibia: Leptodactylidae) from rice agroecosystems, Santa Fe province, Argentina.

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis of vertebrates: Alteration in endogenous retinoids concentration is linked with teratogenic effects. Retinol (ROH), retinoid acid (RA), and isoform 13-Cis-retinoic acid (13-Cis-RA), in plasma of a native adults frog, Leptodactylus chaquensis from a rice field (RF) and a forest (reference site; RS) were measured. ROH did not vary between treatment sites. RA and 13-Cis-RA activities were higher (93.7±8.6 µg mL(-1) and 131.7±11.4 µg mL(-1), respectively) in individuals collected from RF than in those from RS (65.5±8.6 µg mL(-1) and 92.2±10.2 µg mL(-1), respectively). The ratios retinoic acid-retinol (RA/ROH) and 13-Cis-RA/ROH revealed significantly higher values in RF than in RS. RA and 13-Cis-RA concentrations in plasma on wild amphibian's species such as L. chaquensis would be suitable biomarkers of pesticide exposure in field monitoring. Finally, the mechanism of alteration in retinoid metabolites alteration should be further explored both in larvae and adult, considering that the potential exposition and uptake contaminants vary between the double lives of these vertebrates.